JOURNAL OF BACTERIOLOGY, Dec. 1978, p. 1192-1196
Vol. 136, No. 3
0021-9193/78/0136-1192$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Construction of a Hybrid Bactenrophage-Plasmid Recombinant DNA Vector DANIEL J. DONOGHUE* AND PHILLIP A. SHARP Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 11 May 1978
A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 x 10 to 11 x 106 daltons. The colicin El plasmid replicon was fused to the left arn of a Xgt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage X functions or via the colicin El plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, A functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin El plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector. A phage-plasmid hybrid has been previously described (8) which was constructed by cloning the mini-colicin El (mini-ColEl) plasmid (12) in a phage A vector (19). Designated AcollOO, this phage-plasmid hybrid was able to replicate either by the ColEl plasmid replicon or by the phage-encoded functions 0 and P. At 32°C, the thermolabile phage repressor cI857 was functional and lytic phage functions were repressed; thus, XcollOO replicated extrachromosomally as a plasmid, and its DNA could be isolated as covalently closed circular molecules in ethidium bromide-cesium chloride density gradients. However, at the nonpermissive temperature, the phage repressor became inactivated, causing XcollOO to undergo a lytic cycle; when propagated as a phage, XcollOO DNA could be easily isolated by banding virions in CsCl equilbrium gradients. We describe here a deletion derivative of XcollOO which is suitable for use as a recombinant DNA vector. Designated XcollO6B (Fig. ld), this phage-plasmid hybrid vector is analogous in many ways to the Xgt vectors of Thomas et al. (19) and can accommodate EcoRI fragments ranging from 2 x 106 to 11 X 106 daltons. Unlike Agt, however, the ColEl plasmid replicon is present in the phage genome adjacent to gene J. The gene coding for immunity to colicin El protein is also present in XcollO6B and is potentially available as a genetic marker for positive selection. In addition, a fragment of Escherichia coli DNA encoding genes of the arabinose op-
eron was inserted into the central portion of the genome. This provided a convenient genetic marker for the presence or absence of inserted DNA, since the substitution of foreign DNA for the ara fragment resulted in an ara- phage. In addition, the removal of the A-RI-C fragment ensured that only red phage would be present in the phage pool after ligation and transfection; this is important since red+ phage, were they present, would overgrow recombinants containing inserted fragments of foreign DNA (see be-
low). A derivative, AcollO6C (Fig. le) was also constructed carrying the amber mutation SamlOO (10). This mutation is efficiently suppressed by suIII but is not suppressed in celLs which are suor suII. Under these latter conditions, cell lysis fails to occur and larger phage yields can be obtained. AcollOO possesses the end fragments of Agt as well as three inserted EcoRI fragments (Fig. 1), one of which is the mini-ColEl plasmid (12). By applying an ethylenediaminetetraacetate selection protocol (17) to AcollOO, many deletion derivatives could be isolated; one of these, designated AcollO6, had suffered a large deletion, fusing the plasmid replicon to the left EcoRI fragment (Fig. la and b). This was easily documented in EcoRI restriction patterns of these phage DNAs. After digestion with EcoRI, AcollOO yielded a pattern of five restriction fragments (Fig. 2, lane 3) of which the smallest represented the inserted mini-ColEl plasmid; in
1192
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VOL. 136, 1978
Km-ex Mini-ColEI X-RI-C
Left b2
J
a) Xcoi 100-
_
_
_
_
4
4 -
-
-
ori
----_
immL
nt
immi x-RI-C
Right
i c1857 nin5
S+
+ c1857 nin5 S+
b) Xcol 106
A
J ori
C) Xcol 106A
A
J orn imm; X-RI-C + c1857 nin5 SamlOO
d) Xcol 106B
A
J ori immj Ara
J ori
e) Xcol 106C A
immi
Ara
1193
f c1857 nin5 S+ f c1857 nin5 SamIOO
FIG. 1. Genome structure ofphage-plasmid hybrids. Selected A genetic markers are shown in addition to: ori, the origin of replication for the ColEl plasmid replicon; imm, the gene specifying immunity to the colicin El protein; b2, that portion of the b2 region of phage A lying in the left-end EcoRI-A fragment. EcoRI sites are indicated by vertical arrows. (a) The structure of the parent phage AcollOO is shown; the dashed line indicates the deletion which occurred during the generation of AcollO6. (b) AcollO6 was isolated by ethylenediaminetetraacetate selection (17) from AcollOO. (c) AcollO6A was generated by recombination between Agt Saml OO-AC (10) and AcollO6. (d) Acoll06B was constructed by in vitro ligation, using the DNAs of AcollO6 and Agt-ara6 (see text); Agt-ara6 was obtained from M. Thomas and R. W. Davis via D. Botstein. (e) AcollO6C was generated by recombination between Acoll06A and Acoll06B. These maps are not drawn to scale.
12
contrast, AcollO6 yielded only three restriction fragments (Fig. 2, lane 4), representing the two end fragments and the A-RI-C fragment. This digestion pattern was diagnostic of a deletion beginning in the left EcoRI fragment and terminating in the mini-ColEl fragment, thereby fusing a portion of the mini-ColEl fragment into the left end EcoRI-A fragment. A comparison of the genome structures of AgtAC and AcollO6 shows that they are very similar: each phage possesses two EcoRI cleavage sites, and each contains the A-RI-C fragment in the center portion of the genome. However, Agt-AC retains a portion of the nonessential b2 region lying between gene J and the EcoRI cleavage site. In AcollO6, the portion of the b2 region FIG. 2. 1EcoRI cleavage patterns ofphage-plasmid contained in the left-end EcoRI-A fragment was hybrids. Lcmne 1, A DNA size markers, with the follow- replaced by a portion of the mini-ColEl plasmid, ing molecuilar sizes (in megadaltons): (A) 13.7; (D) while keeping the overall size of this fragment 4.74; (E) 3. 73; (C) 3.48; (B) 3&02; (F) 2.13 (20). Lane Z, approximately the same. DNA ofplczsmidpML21 (15), showing the mini-ColE) T The red gene Of A, which affects general refriagment (designated by arrow) of 2.32 x 1(9 daltons. Lane 3, AcollOO DNA, yielding five EcoRI cleavage combination and the growth of A on various fragments, ,of which the smallest is the mini-ColE) hosts (4,9),maps on the A-RI-C fragment. Phage fragment (Idesignated by arrow). The molecular sizes which are red+ tend to overgrow phage which of the five fragments are (in megadaltons): left end, are red; for this reason, many phage vectors 13.7; right end, 8.9; A-RI-C, 3.48; Km-ex, 3.02; mini- have been specifically constructed so as to reColE1, 2.3 2 (7). Lane 4, Acol1O6 DNA. Of three in- move this fragment and substitute in its place serted fragments in AcollOO, only the A-RI-C fragment some other fragment of DNA. A fragment of the remained (designated by arrow). A portion of the E. coli arabinose operon, already cloned into . ColE) plasrmid was fused into the left-end fragment, . a but this di d not alter its mobility. Lane 5, Acoll06B Agt, provided anIdeal candidate with which to DNA. The A-RI-C fragment was removed and re- replace the A-RI-C fragment of AcollO6. Approxplaced witA the ara fragment (designated by arrow). imately 3.5 x 106 daltons in size, this ara fragThe A-RI-(C fragment and the ara fragment are the ment permits phage to be easily scored on arasame size and have identical mobilities in this gel binose-tetrazolium dye plates with appropriate
x
3
4
5
1194
NOTES
ara indicator cells (14). The DNAs of XcollO6 and Xgt-ara were sequentially treated with EcoRI and T4 DNA ligase and then transfected into E. coli C600 rk mk . The phage designated XcollO6B was isolated from this reaction and contains the ara fragment of DNA in place of the A-RI-C fragment (Fig. ld). Because the ara fragment is approximately the same size as the A-RI-C fragment, the EcoRI digestion pattern of XcollO6B is indistinguishable from that of its parent AcollO6 (cf. lanes 4 and 5 of Fig. 2). The presence of the ara fragment in XcollO6B can be physically confirmed, however, by the presence of a new KpnI restriction endonuclease cleavage site which is absent in the A-RI-C fragment (data not shown) or, more directly, by electron microscopy. Figures 3A and B present a heteroduplex between the DNAs of Agt-ara and XcollO6B. These DNAs are homologous everywhere except for a small substitution bubble in the central portion of the genome. The longer arm of this substitution loop represents that portion of the b2 region present in the left-end EcoRI-A fragment of Agtara which was deleted from XcollO6B. The shorter arm in the substitution bubble represents DNA from the ColEl plasmid which was inserted into AcollO6B. Figure 3C presents a heteroduplex between the DNA of AcollO6B and that of its parent XcollOO. These two phages both contain the ColEl plasmid replicon, represented by the duplex region in the center of the molecule. The large single-stranded loop represents the DNA which was deleted from XcollOO during the generation of XcollO6. In the same heteroduplex, the large substitution loop shows where the A-RI-C fragment of XcollOO was replaced with the ara fragment of AcollO6B. Measurements of such heteroduplexes show that the left-end fragment of AcollO6B is only 0.3 x 106 daltons smaller than that of Agt. Thus, XcollO6B should accommodate the same size range of EcoRI fragments as does Xgt (about 2 x 106 to 11 x 106 daltons). Like Agt, the two end fragments of XcollO6B are by themselves too small to be packaged efficiently. Since an inserted fragment of DNA is required for efficient packaging, this provides a positive selection for the growth of recombinants forned during in vitro ligation reactions (19). It was important to demonstrate that, both in the absence and in the presence of chloramphenicol, the phage-plasmid hybrid could replicate in the manner anticipated for a CoJEl derivative. The ColEl plasmid (ca. 4.2 x 106 daltons) is maintained at a level of 10 to 15 copies per chromosome; if XcollO6B were maintained as a plasmid at the same level, then based on its
J. BACTERIOL.
FIG. 3 Elctron microscopi analysis of phageplasmid hybrids. (A) Heteroduplex of Acol106B with Agt-ara. The DNA8 are homologous everywhere except for a substitution bubble in the central portion of the molecul. The bar represents 10 daltons. (B) Higher magnification of the heteroduplex in (A). The longer arm of the substitution loop represents that portion of the Ab2 region present in Agt-ara but deleted in Aco11O6B, and measured (1.48 ± 0.12) x 106f dalton.s. The shorter arm of the substitution loop represents that portion of the ColEl plasmid fused into Acol106B, and measured (1.21 ± 0.08) x 106 dalton.. The bar represents 106 dalton. of duplex DNA. (C) Heteroduplex of XcollO6B with its parent Acol1lX). The large single-stranded DNA loop represents the spontaneous deletion in ecollO( which gave rise to Xco:1O6; this deletion loop measured (5.5 ± 0.2) x 106 dalton.. The intervening region of doublestranded DNA represents that portion of the miniColE) plasmid common to both DNAs; this segment measured (1.38 ± 0,08) x 106 dalton.. The two arms of the large sub-stitution loop represent the -RI-C ara fragment in fragment in XcollOO and theXcoX*06B and had indistinguishable measurements. The ,-RIC fr-agment has been previously measured as 3.48 x 106 dalton. (20). The magnification in (C) is the -same as in (B) above. The bar represents 106 dalton. of duplex DNA.
larger size (26 x 106 daltons) one would predict a copy number of two to three copies per chromosome (1, 11).
VOL. 136, 1978
To physically measure the copy number, the phage-plasmid hybrid was introduced into AB2495, a thymine-requiring strain. A culture of AB2495(XcollO6B) was then labeled with [3H]thymine at 320C, after which the total DNA was extracted and banded in ethidium bromidecesium chloride density gradients. A small peak of radioactivity was seen in the form I position, representing covalently closed circular DNA, compared with a much larger peak of radioactivity in the upper band position representing cellular DNA (Fig. 4A). The radioactivity present in the form I peak accounted for 2.2% of the total radioactivity present in these two peaks; taking the E. coli chromosome to be 2.5 x 109 daltons (21) and knowing the molecular weight of XcollO6B, then this form I radioactivity corresponds to about 2.1 copies of XcollO6 per chromosome. Because some XcollO6B DNA may have been present as relaxed, circular, form II DNA, this number represents a minimal estimate of its copy number. Nonetheless, this estimate is consistent with the copy number predicted above. Plasmid ColEl and its derivatives replicate in the presence of chloramphenicol, even though cellular DNA synthesis ceases rapidly; eventually, the plasmid DNA may represent as much as 45% of the total DNA in the cell (6, 11). When AB2495(Acol1O6B) was uniformly labeled with [3H]thymine and then incubated for 16 h in the presence of chloramphenicol, the ColEl plasmid replicon was able to direct replication of the phage-plasmid hybrid. After chloramphenicol amplification, 40% of the total radioactivity was recovered in the form I position of the gradient (Fig. 4B). These data indicate that XcollO6B is maintained extrachromosomally, but they do not directly rule out the existence of additional copies integrated into the bacterial chromosome. This latter possibility seems unlikely, however, given that XcollO6B is deleted for all X site-specific recombination functions. In the case of the parent phage XcollOO, which carries X site-specific recombination functions as well as the ColEl plasmid replicon, it seems probable that the phage would exist both extrachromosomally and also integrated into the chromosome; this would be true unless the plasmid replicon in some fashion interfered with A site-specific recombination. Most phage vectors to date have been derivatives of phage A (2, 16, 18, 19). With these vectors, recombinants are generally formed by the substitution of foreign DNA in place of nonessential A genetic information. Most X recombinants are unable to form stable lysogens and must be propagated lytically; this can be due
NOTES
1195
-0 c) cL
C 0 c
I.
.a_ 0
30- B -r tn
20E
A *
10
10
20
30
Fraction number FIG. 4. Acol106B replicates as a covalently closed circular DNA molecule. (A) In the absence of chloramphenicol: AB2495(AcollO6B) was uniformly labeled with [3H]thymine and prepared for ethidium bromide-cesium chloride density gradient centrifugation by the method of Womble et al. (21). The radioactivity present in the form Iposition (shown on expanded scale in inset) represents about 2.2% of the radioactivity present in the upper band plus form I band peaks. (B) In the presence of chloramphenicol: AB2495(AcollO6B) was uniformly labeled with [3H]thymine, treated with chloramphenicol (20 Pg/ml) for 16 h and then prepared for ethidium bromide-cesium chloride density gradient centrifugation, as described for (A). The radioactivity in the form I position now accounts for 40% of the total radioactivity in the upper band plus form I band peaks. The form I position was n = 1.3914 (1.612 g/ml), and the upper band position was n - 1.3885 (1.577 g/ml). Data are presented as percentages of the total incorporated radioactivity, which was 1.0 x 105 cpm for the portions counted for (A) and 2.1 x 1ltf cpm for those counted for (B).
either to deletion of the att-int-xis region or to inactivation of the phage repressor gene. Packaging restrictions limit the amount of DNA which can be inserted to about 11 x 106 daltons, but also provide a positive selection for recombinants by making the vector DNA alone too small to be efficiently encapsidated. Another characteristic of phage vectors is that they permit cloned fragments of DNA to be readily transferred between different strains of bacteria
1196
NOTES
as phage without a need for DNA transfections. In contrast, most plasmid vectors have been derived either from ColEl or from various Rfactors (3, 5, 13). With these vectors, the cloned fragment is stably maintained in the bacterium at a copy number determined by the specific plasmid replicon and the size of the fragment; as a result, plasmid vectors tend to place few limitations on the size of the DNA fragment which can be cloned. Unlike phage, however, most plasmids can be moved between different bacterial strains only by DNA transfection. In certain experimental designs, the dual phage-plasmid characteristics of XcollO6B may provide an attractive alternative to other vector systems. Most phage vectors previously constructed have been difficult to integrate stably into the host chromosome. Deleted either for site-specific integration functions or else lacking a functional repressor, genetic selections by complementation have required double lysogen formation with a helper phage. In contrast, at 32°C XcollO6B is stably maintained as an extrachromosomal element because of the ColEl plasmid replicon and at a higher copy number than if integration and lysogeny had occurred via normal phage functions. In addition, the DNA of XcollO6B can be prepared either as linear phage DNA or as covalently closed circular plasmid DNA. We thank Ellen Rothenberg, Arnold Berk, and David Steffen for stimulating discussions, and also Robert Schleif and David Botstein for providing necessary strains. D.J.D. was supported by a fellowship from the Whitaker Health Sciences Fund. P.A.S. gratefully acknowledges a grant (no. VC-151A) and a Career Development Award from the American Cancer Society and a Cancer Center Core Grant (no. CA-14951).
LITERATURE CITED 1. Bazaral, M., and D. R. Helinski. 1970. Replication of a bacterial plasmid and an episome in Escherichia coli.
Biochemistry 9:399-406. 2. Blattner, F. R., B. G. Williams, A. E. Blechl, K. Denniston-Thompson, H. E. Farber, L. Furlong, D. J. Grunwald, D. 0. Kiefer, D. D. Moore, J. W. Schumm, E. L. Sheldon, and 0. Smithies. 1977. Charon phages: safer derivatives of phage lambda for DNA cloning. Science 196:161-169. 3. Boyer, H. W., M. Betlach, F. Bolivar, R. L. Rodriguez, H. L. Heyneker, J. Shine, and H. M. Goodman. 1977. The construction of molecular cloning vehicles, p. 9-27. In R. F. Beers, Jr., and E. G. Bassett (ed.), Recombinant molecules: impact on science and society. Raven Press, New York. 4. Cameron, J. R., S. M. Panasenko, I. R. Lehman, and R. W. Davis. 1975. In vitro construction of bacteriophage A carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for
J. BACTERIOL. DNA ligase. Proc. Natl. Acad. Sci. U.S.A. 72:3416-3420. 5. Chang, A. C. Y., and S. N. Cohen. 1974. Genome construction between bacterial species in vitro: replication and expression of Staphylococcus plasmid genes in Escherchia coli. Proc. Natl. Acad. Sci. U.S.A. 71: 1030-1034. 6. Clewell, D. B. 1972. Nature of Col El plasmid replication in Escherichia coli in the presence of chloramphenicol. J. Bacteriol. 110:667-676. 7. Donoghue, D. J., and P. A. Sharp. 1977. Model recombinants for the development and manipulation of EK2 phage vector systems, p. 41-53. In G. Wilcox, J. Abelson, and C. F. Fox (ed.), Eucaryotic genetic systems: ICNUCLA symposia on molecular and cellular biology, vol. 8. Academic Press, Inc., New York. 8. Donoghue, D. J., and P. A. Sharp. 1978. Replication of colicin El plasmid DNA in vivo requires no plasmidencoded proteins. J. Bacteriol. 133:1287-1294. 9. Enquist, L. W., and A. Skalka. 1973. Replication of bacteriophage A DNA dependent on the function of host and viral genes. J. Mol. Biol. 75:185-212. 10. Enquist, L. W., D. Tiemeier, P. Leder, R. Weisberg, and N. Sternberg. 1976. Safer derivatives of bacteriophage Agt-AC for use in the cloning of recombinant DNA molecules. Nature (London) 259:596-598. 11. Helinski, D. R., M. A. Lovett, P. H. Wiliams, L. Katz, J. Collins, Y. Kuperstoch-Portnoy, S. Sato, R. W. Leavitt, R. Sparks, V. Hershfield, D. G. Guiney, and D. G. Blair. 1975. Modes of plasmid DNA replication in Escherichia coli, p. 514-536. In M. Goulian and P. Hanawalt (ed.), DNA synthesis and its regulation. W. A. Benjamin, Inc., Reading, Mass. 12. Hershfield, V., H. W. Boyer, L. Chow, and D. R. Helinski. 1976. Characterization of a mini-ColEl plasmid. J. Bacteriol. 126:447-453. 13. Hershfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett, and D. R. Helinski. 1974. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc. Natl. Acad. Sci. U.S.A. 71:3455-3459. 14. Lis, J. T., and R. Schleif. 1975. The isolation and characterization of plaque-forming arabinose transducing bacteriophage A. J. Mol. Biol. 95:395-407. 15. Lovett, M. A., and D. R. Helinski. 1976. Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'km plasmid. J. Bacteriol. 127: 982-987. 16. Murray, N. E., and K. Murray. 1974. Manipulation of restriction targets in phage A to form receptor chromosomes for DNA fragments. Nature (London) 251: 476-481. 17. Parkinson, J. S., and R. J. Huskey. 1971. Deletion mutants of bacteriophage lambda. I. Isolation and initial characterization. J. Mol. Biol. 56:369-384. 18. Rambach, A., and P. Tiollais. 1974. Bacteriophage A having EcoRI endonuclease sites only in the nonessential region of the genome. Proc. Natl. Acad. Sci. U.S.A. 71:3927-3930. 19. Thomas, M., J. R. Cameron, and R. W. Davis. 1974. Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA. Proc. Natl. Acad. Sci. U.S.A. 71: 4579-4583. 20. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. 21. Womble, D. D., D. P. Taylor, and R. H. Rownd. 1977. Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation. J. Bacteriol. 130:148-153.
Vol. 136, No. 3
0021-9193/78/0136-1192$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Construction of a Hybrid Bactenrophage-Plasmid Recombinant DNA Vector DANIEL J. DONOGHUE* AND PHILLIP A. SHARP Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received for publication 11 May 1978
A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 x 10 to 11 x 106 daltons. The colicin El plasmid replicon was fused to the left arn of a Xgt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage X functions or via the colicin El plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, A functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin El plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector. A phage-plasmid hybrid has been previously described (8) which was constructed by cloning the mini-colicin El (mini-ColEl) plasmid (12) in a phage A vector (19). Designated AcollOO, this phage-plasmid hybrid was able to replicate either by the ColEl plasmid replicon or by the phage-encoded functions 0 and P. At 32°C, the thermolabile phage repressor cI857 was functional and lytic phage functions were repressed; thus, XcollOO replicated extrachromosomally as a plasmid, and its DNA could be isolated as covalently closed circular molecules in ethidium bromide-cesium chloride density gradients. However, at the nonpermissive temperature, the phage repressor became inactivated, causing XcollOO to undergo a lytic cycle; when propagated as a phage, XcollOO DNA could be easily isolated by banding virions in CsCl equilbrium gradients. We describe here a deletion derivative of XcollOO which is suitable for use as a recombinant DNA vector. Designated XcollO6B (Fig. ld), this phage-plasmid hybrid vector is analogous in many ways to the Xgt vectors of Thomas et al. (19) and can accommodate EcoRI fragments ranging from 2 x 106 to 11 X 106 daltons. Unlike Agt, however, the ColEl plasmid replicon is present in the phage genome adjacent to gene J. The gene coding for immunity to colicin El protein is also present in XcollO6B and is potentially available as a genetic marker for positive selection. In addition, a fragment of Escherichia coli DNA encoding genes of the arabinose op-
eron was inserted into the central portion of the genome. This provided a convenient genetic marker for the presence or absence of inserted DNA, since the substitution of foreign DNA for the ara fragment resulted in an ara- phage. In addition, the removal of the A-RI-C fragment ensured that only red phage would be present in the phage pool after ligation and transfection; this is important since red+ phage, were they present, would overgrow recombinants containing inserted fragments of foreign DNA (see be-
low). A derivative, AcollO6C (Fig. le) was also constructed carrying the amber mutation SamlOO (10). This mutation is efficiently suppressed by suIII but is not suppressed in celLs which are suor suII. Under these latter conditions, cell lysis fails to occur and larger phage yields can be obtained. AcollOO possesses the end fragments of Agt as well as three inserted EcoRI fragments (Fig. 1), one of which is the mini-ColEl plasmid (12). By applying an ethylenediaminetetraacetate selection protocol (17) to AcollOO, many deletion derivatives could be isolated; one of these, designated AcollO6, had suffered a large deletion, fusing the plasmid replicon to the left EcoRI fragment (Fig. la and b). This was easily documented in EcoRI restriction patterns of these phage DNAs. After digestion with EcoRI, AcollOO yielded a pattern of five restriction fragments (Fig. 2, lane 3) of which the smallest represented the inserted mini-ColEl plasmid; in
1192
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VOL. 136, 1978
Km-ex Mini-ColEI X-RI-C
Left b2
J
a) Xcoi 100-
_
_
_
_
4
4 -
-
-
ori
----_
immL
nt
immi x-RI-C
Right
i c1857 nin5
S+
+ c1857 nin5 S+
b) Xcol 106
A
J ori
C) Xcol 106A
A
J orn imm; X-RI-C + c1857 nin5 SamlOO
d) Xcol 106B
A
J ori immj Ara
J ori
e) Xcol 106C A
immi
Ara
1193
f c1857 nin5 S+ f c1857 nin5 SamIOO
FIG. 1. Genome structure ofphage-plasmid hybrids. Selected A genetic markers are shown in addition to: ori, the origin of replication for the ColEl plasmid replicon; imm, the gene specifying immunity to the colicin El protein; b2, that portion of the b2 region of phage A lying in the left-end EcoRI-A fragment. EcoRI sites are indicated by vertical arrows. (a) The structure of the parent phage AcollOO is shown; the dashed line indicates the deletion which occurred during the generation of AcollO6. (b) AcollO6 was isolated by ethylenediaminetetraacetate selection (17) from AcollOO. (c) AcollO6A was generated by recombination between Agt Saml OO-AC (10) and AcollO6. (d) Acoll06B was constructed by in vitro ligation, using the DNAs of AcollO6 and Agt-ara6 (see text); Agt-ara6 was obtained from M. Thomas and R. W. Davis via D. Botstein. (e) AcollO6C was generated by recombination between Acoll06A and Acoll06B. These maps are not drawn to scale.
12
contrast, AcollO6 yielded only three restriction fragments (Fig. 2, lane 4), representing the two end fragments and the A-RI-C fragment. This digestion pattern was diagnostic of a deletion beginning in the left EcoRI fragment and terminating in the mini-ColEl fragment, thereby fusing a portion of the mini-ColEl fragment into the left end EcoRI-A fragment. A comparison of the genome structures of AgtAC and AcollO6 shows that they are very similar: each phage possesses two EcoRI cleavage sites, and each contains the A-RI-C fragment in the center portion of the genome. However, Agt-AC retains a portion of the nonessential b2 region lying between gene J and the EcoRI cleavage site. In AcollO6, the portion of the b2 region FIG. 2. 1EcoRI cleavage patterns ofphage-plasmid contained in the left-end EcoRI-A fragment was hybrids. Lcmne 1, A DNA size markers, with the follow- replaced by a portion of the mini-ColEl plasmid, ing molecuilar sizes (in megadaltons): (A) 13.7; (D) while keeping the overall size of this fragment 4.74; (E) 3. 73; (C) 3.48; (B) 3&02; (F) 2.13 (20). Lane Z, approximately the same. DNA ofplczsmidpML21 (15), showing the mini-ColE) T The red gene Of A, which affects general refriagment (designated by arrow) of 2.32 x 1(9 daltons. Lane 3, AcollOO DNA, yielding five EcoRI cleavage combination and the growth of A on various fragments, ,of which the smallest is the mini-ColE) hosts (4,9),maps on the A-RI-C fragment. Phage fragment (Idesignated by arrow). The molecular sizes which are red+ tend to overgrow phage which of the five fragments are (in megadaltons): left end, are red; for this reason, many phage vectors 13.7; right end, 8.9; A-RI-C, 3.48; Km-ex, 3.02; mini- have been specifically constructed so as to reColE1, 2.3 2 (7). Lane 4, Acol1O6 DNA. Of three in- move this fragment and substitute in its place serted fragments in AcollOO, only the A-RI-C fragment some other fragment of DNA. A fragment of the remained (designated by arrow). A portion of the E. coli arabinose operon, already cloned into . ColE) plasrmid was fused into the left-end fragment, . a but this di d not alter its mobility. Lane 5, Acoll06B Agt, provided anIdeal candidate with which to DNA. The A-RI-C fragment was removed and re- replace the A-RI-C fragment of AcollO6. Approxplaced witA the ara fragment (designated by arrow). imately 3.5 x 106 daltons in size, this ara fragThe A-RI-(C fragment and the ara fragment are the ment permits phage to be easily scored on arasame size and have identical mobilities in this gel binose-tetrazolium dye plates with appropriate
x
3
4
5
1194
NOTES
ara indicator cells (14). The DNAs of XcollO6 and Xgt-ara were sequentially treated with EcoRI and T4 DNA ligase and then transfected into E. coli C600 rk mk . The phage designated XcollO6B was isolated from this reaction and contains the ara fragment of DNA in place of the A-RI-C fragment (Fig. ld). Because the ara fragment is approximately the same size as the A-RI-C fragment, the EcoRI digestion pattern of XcollO6B is indistinguishable from that of its parent AcollO6 (cf. lanes 4 and 5 of Fig. 2). The presence of the ara fragment in XcollO6B can be physically confirmed, however, by the presence of a new KpnI restriction endonuclease cleavage site which is absent in the A-RI-C fragment (data not shown) or, more directly, by electron microscopy. Figures 3A and B present a heteroduplex between the DNAs of Agt-ara and XcollO6B. These DNAs are homologous everywhere except for a small substitution bubble in the central portion of the genome. The longer arm of this substitution loop represents that portion of the b2 region present in the left-end EcoRI-A fragment of Agtara which was deleted from XcollO6B. The shorter arm in the substitution bubble represents DNA from the ColEl plasmid which was inserted into AcollO6B. Figure 3C presents a heteroduplex between the DNA of AcollO6B and that of its parent XcollOO. These two phages both contain the ColEl plasmid replicon, represented by the duplex region in the center of the molecule. The large single-stranded loop represents the DNA which was deleted from XcollOO during the generation of XcollO6. In the same heteroduplex, the large substitution loop shows where the A-RI-C fragment of XcollOO was replaced with the ara fragment of AcollO6B. Measurements of such heteroduplexes show that the left-end fragment of AcollO6B is only 0.3 x 106 daltons smaller than that of Agt. Thus, XcollO6B should accommodate the same size range of EcoRI fragments as does Xgt (about 2 x 106 to 11 x 106 daltons). Like Agt, the two end fragments of XcollO6B are by themselves too small to be packaged efficiently. Since an inserted fragment of DNA is required for efficient packaging, this provides a positive selection for the growth of recombinants forned during in vitro ligation reactions (19). It was important to demonstrate that, both in the absence and in the presence of chloramphenicol, the phage-plasmid hybrid could replicate in the manner anticipated for a CoJEl derivative. The ColEl plasmid (ca. 4.2 x 106 daltons) is maintained at a level of 10 to 15 copies per chromosome; if XcollO6B were maintained as a plasmid at the same level, then based on its
J. BACTERIOL.
FIG. 3 Elctron microscopi analysis of phageplasmid hybrids. (A) Heteroduplex of Acol106B with Agt-ara. The DNA8 are homologous everywhere except for a substitution bubble in the central portion of the molecul. The bar represents 10 daltons. (B) Higher magnification of the heteroduplex in (A). The longer arm of the substitution loop represents that portion of the Ab2 region present in Agt-ara but deleted in Aco11O6B, and measured (1.48 ± 0.12) x 106f dalton.s. The shorter arm of the substitution loop represents that portion of the ColEl plasmid fused into Acol106B, and measured (1.21 ± 0.08) x 106 dalton.. The bar represents 106 dalton. of duplex DNA. (C) Heteroduplex of XcollO6B with its parent Acol1lX). The large single-stranded DNA loop represents the spontaneous deletion in ecollO( which gave rise to Xco:1O6; this deletion loop measured (5.5 ± 0.2) x 106 dalton.. The intervening region of doublestranded DNA represents that portion of the miniColE) plasmid common to both DNAs; this segment measured (1.38 ± 0,08) x 106 dalton.. The two arms of the large sub-stitution loop represent the -RI-C ara fragment in fragment in XcollOO and theXcoX*06B and had indistinguishable measurements. The ,-RIC fr-agment has been previously measured as 3.48 x 106 dalton. (20). The magnification in (C) is the -same as in (B) above. The bar represents 106 dalton. of duplex DNA.
larger size (26 x 106 daltons) one would predict a copy number of two to three copies per chromosome (1, 11).
VOL. 136, 1978
To physically measure the copy number, the phage-plasmid hybrid was introduced into AB2495, a thymine-requiring strain. A culture of AB2495(XcollO6B) was then labeled with [3H]thymine at 320C, after which the total DNA was extracted and banded in ethidium bromidecesium chloride density gradients. A small peak of radioactivity was seen in the form I position, representing covalently closed circular DNA, compared with a much larger peak of radioactivity in the upper band position representing cellular DNA (Fig. 4A). The radioactivity present in the form I peak accounted for 2.2% of the total radioactivity present in these two peaks; taking the E. coli chromosome to be 2.5 x 109 daltons (21) and knowing the molecular weight of XcollO6B, then this form I radioactivity corresponds to about 2.1 copies of XcollO6 per chromosome. Because some XcollO6B DNA may have been present as relaxed, circular, form II DNA, this number represents a minimal estimate of its copy number. Nonetheless, this estimate is consistent with the copy number predicted above. Plasmid ColEl and its derivatives replicate in the presence of chloramphenicol, even though cellular DNA synthesis ceases rapidly; eventually, the plasmid DNA may represent as much as 45% of the total DNA in the cell (6, 11). When AB2495(Acol1O6B) was uniformly labeled with [3H]thymine and then incubated for 16 h in the presence of chloramphenicol, the ColEl plasmid replicon was able to direct replication of the phage-plasmid hybrid. After chloramphenicol amplification, 40% of the total radioactivity was recovered in the form I position of the gradient (Fig. 4B). These data indicate that XcollO6B is maintained extrachromosomally, but they do not directly rule out the existence of additional copies integrated into the bacterial chromosome. This latter possibility seems unlikely, however, given that XcollO6B is deleted for all X site-specific recombination functions. In the case of the parent phage XcollOO, which carries X site-specific recombination functions as well as the ColEl plasmid replicon, it seems probable that the phage would exist both extrachromosomally and also integrated into the chromosome; this would be true unless the plasmid replicon in some fashion interfered with A site-specific recombination. Most phage vectors to date have been derivatives of phage A (2, 16, 18, 19). With these vectors, recombinants are generally formed by the substitution of foreign DNA in place of nonessential A genetic information. Most X recombinants are unable to form stable lysogens and must be propagated lytically; this can be due
NOTES
1195
-0 c) cL
C 0 c
I.
.a_ 0
30- B -r tn
20E
A *
10
10
20
30
Fraction number FIG. 4. Acol106B replicates as a covalently closed circular DNA molecule. (A) In the absence of chloramphenicol: AB2495(AcollO6B) was uniformly labeled with [3H]thymine and prepared for ethidium bromide-cesium chloride density gradient centrifugation by the method of Womble et al. (21). The radioactivity present in the form Iposition (shown on expanded scale in inset) represents about 2.2% of the radioactivity present in the upper band plus form I band peaks. (B) In the presence of chloramphenicol: AB2495(AcollO6B) was uniformly labeled with [3H]thymine, treated with chloramphenicol (20 Pg/ml) for 16 h and then prepared for ethidium bromide-cesium chloride density gradient centrifugation, as described for (A). The radioactivity in the form I position now accounts for 40% of the total radioactivity in the upper band plus form I band peaks. The form I position was n = 1.3914 (1.612 g/ml), and the upper band position was n - 1.3885 (1.577 g/ml). Data are presented as percentages of the total incorporated radioactivity, which was 1.0 x 105 cpm for the portions counted for (A) and 2.1 x 1ltf cpm for those counted for (B).
either to deletion of the att-int-xis region or to inactivation of the phage repressor gene. Packaging restrictions limit the amount of DNA which can be inserted to about 11 x 106 daltons, but also provide a positive selection for recombinants by making the vector DNA alone too small to be efficiently encapsidated. Another characteristic of phage vectors is that they permit cloned fragments of DNA to be readily transferred between different strains of bacteria
1196
NOTES
as phage without a need for DNA transfections. In contrast, most plasmid vectors have been derived either from ColEl or from various Rfactors (3, 5, 13). With these vectors, the cloned fragment is stably maintained in the bacterium at a copy number determined by the specific plasmid replicon and the size of the fragment; as a result, plasmid vectors tend to place few limitations on the size of the DNA fragment which can be cloned. Unlike phage, however, most plasmids can be moved between different bacterial strains only by DNA transfection. In certain experimental designs, the dual phage-plasmid characteristics of XcollO6B may provide an attractive alternative to other vector systems. Most phage vectors previously constructed have been difficult to integrate stably into the host chromosome. Deleted either for site-specific integration functions or else lacking a functional repressor, genetic selections by complementation have required double lysogen formation with a helper phage. In contrast, at 32°C XcollO6B is stably maintained as an extrachromosomal element because of the ColEl plasmid replicon and at a higher copy number than if integration and lysogeny had occurred via normal phage functions. In addition, the DNA of XcollO6B can be prepared either as linear phage DNA or as covalently closed circular plasmid DNA. We thank Ellen Rothenberg, Arnold Berk, and David Steffen for stimulating discussions, and also Robert Schleif and David Botstein for providing necessary strains. D.J.D. was supported by a fellowship from the Whitaker Health Sciences Fund. P.A.S. gratefully acknowledges a grant (no. VC-151A) and a Career Development Award from the American Cancer Society and a Cancer Center Core Grant (no. CA-14951).
LITERATURE CITED 1. Bazaral, M., and D. R. Helinski. 1970. Replication of a bacterial plasmid and an episome in Escherichia coli.
Biochemistry 9:399-406. 2. Blattner, F. R., B. G. Williams, A. E. Blechl, K. Denniston-Thompson, H. E. Farber, L. Furlong, D. J. Grunwald, D. 0. Kiefer, D. D. Moore, J. W. Schumm, E. L. Sheldon, and 0. Smithies. 1977. Charon phages: safer derivatives of phage lambda for DNA cloning. Science 196:161-169. 3. Boyer, H. W., M. Betlach, F. Bolivar, R. L. Rodriguez, H. L. Heyneker, J. Shine, and H. M. Goodman. 1977. The construction of molecular cloning vehicles, p. 9-27. In R. F. Beers, Jr., and E. G. Bassett (ed.), Recombinant molecules: impact on science and society. Raven Press, New York. 4. Cameron, J. R., S. M. Panasenko, I. R. Lehman, and R. W. Davis. 1975. In vitro construction of bacteriophage A carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for
J. BACTERIOL. DNA ligase. Proc. Natl. Acad. Sci. U.S.A. 72:3416-3420. 5. Chang, A. C. Y., and S. N. Cohen. 1974. Genome construction between bacterial species in vitro: replication and expression of Staphylococcus plasmid genes in Escherchia coli. Proc. Natl. Acad. Sci. U.S.A. 71: 1030-1034. 6. Clewell, D. B. 1972. Nature of Col El plasmid replication in Escherichia coli in the presence of chloramphenicol. J. Bacteriol. 110:667-676. 7. Donoghue, D. J., and P. A. Sharp. 1977. Model recombinants for the development and manipulation of EK2 phage vector systems, p. 41-53. In G. Wilcox, J. Abelson, and C. F. Fox (ed.), Eucaryotic genetic systems: ICNUCLA symposia on molecular and cellular biology, vol. 8. Academic Press, Inc., New York. 8. Donoghue, D. J., and P. A. Sharp. 1978. Replication of colicin El plasmid DNA in vivo requires no plasmidencoded proteins. J. Bacteriol. 133:1287-1294. 9. Enquist, L. W., and A. Skalka. 1973. Replication of bacteriophage A DNA dependent on the function of host and viral genes. J. Mol. Biol. 75:185-212. 10. Enquist, L. W., D. Tiemeier, P. Leder, R. Weisberg, and N. Sternberg. 1976. Safer derivatives of bacteriophage Agt-AC for use in the cloning of recombinant DNA molecules. Nature (London) 259:596-598. 11. Helinski, D. R., M. A. Lovett, P. H. Wiliams, L. Katz, J. Collins, Y. Kuperstoch-Portnoy, S. Sato, R. W. Leavitt, R. Sparks, V. Hershfield, D. G. Guiney, and D. G. Blair. 1975. Modes of plasmid DNA replication in Escherichia coli, p. 514-536. In M. Goulian and P. Hanawalt (ed.), DNA synthesis and its regulation. W. A. Benjamin, Inc., Reading, Mass. 12. Hershfield, V., H. W. Boyer, L. Chow, and D. R. Helinski. 1976. Characterization of a mini-ColEl plasmid. J. Bacteriol. 126:447-453. 13. Hershfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett, and D. R. Helinski. 1974. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc. Natl. Acad. Sci. U.S.A. 71:3455-3459. 14. Lis, J. T., and R. Schleif. 1975. The isolation and characterization of plaque-forming arabinose transducing bacteriophage A. J. Mol. Biol. 95:395-407. 15. Lovett, M. A., and D. R. Helinski. 1976. Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'km plasmid. J. Bacteriol. 127: 982-987. 16. Murray, N. E., and K. Murray. 1974. Manipulation of restriction targets in phage A to form receptor chromosomes for DNA fragments. Nature (London) 251: 476-481. 17. Parkinson, J. S., and R. J. Huskey. 1971. Deletion mutants of bacteriophage lambda. I. Isolation and initial characterization. J. Mol. Biol. 56:369-384. 18. Rambach, A., and P. Tiollais. 1974. Bacteriophage A having EcoRI endonuclease sites only in the nonessential region of the genome. Proc. Natl. Acad. Sci. U.S.A. 71:3927-3930. 19. Thomas, M., J. R. Cameron, and R. W. Davis. 1974. Viable molecular hybrids of bacteriophage lambda and eukaryotic DNA. Proc. Natl. Acad. Sci. U.S.A. 71: 4579-4583. 20. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease. J. Mol. Biol. 91:315-328. 21. Womble, D. D., D. P. Taylor, and R. H. Rownd. 1977. Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation. J. Bacteriol. 130:148-153.
